Flow Cytometry

Flow cytometry is a powerful analytical technique used to count, examine, and sort microscopic particles, such as cells,vsuspended in a fluid stream. As the particles pass through a hydrodynamically focused flow, they intersect a laser beam of a single wavelength, allowing their physical and chemical characteristics to be measured in real time.

Light signals generated during this interaction are collected by multiple detectors. Forward Scatter (FSC), detected in line with the laser, correlates with cell size, while Side Scatter (SSC), detected at a 90° angle, reflects internal complexity such as nuclear shape, cytoplasmic granularity, and membrane structure. Additional detectors capture fluorescence emitted from labeled molecules within or on the surface of the particles.

By analysing fluctuations in scattered and fluorescent light, detailed information about each individual particle can be obtained. Fluorescence-activated cell sorting (FACS) is a specialised application of flow cytometry that enables the physical separation of cells based on predefined criteria. In FACS, the fluid stream is vibrated to form individual droplets, each containing a single particle. Droplets meeting the selected scatter and fluorescence parameters are electrically charged and deflected by an electrostatic field into separate collection vessels.

Cell surface staining can be performed using either direct or indirect immunofluorescence methods. In direct staining, cells are incubated with fluorochrome-conjugated antibodies (e.g. FITC), offering a faster protocol with reduced risk of non-specific binding. Indirect staining involves incubation with an unconjugated primary antibody, followed by a fluorochrome-labelled secondary antibody, allowing greater signal amplification and flexibility in fluorochrome selection (e.g. FITC, PE, Cy5).

Successful flow cytometry experiments rely on careful optimisation of experimental conditions, including antibody titration, appropriate controls for accurate instrument setup, and optimised fixation and permeabilisation protocols where required.

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Advanced flow cytometry services for accurate cell analysis and sorting.

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